Agonist-stimulated [35S]GTPgammaS binding allows the visualization of receptor-activated G-proteins, thus revealing the anatomical localization of functional receptor activity. In the present study, agonist-stimulated [35S]GTPgammaS binding was used to demonstrate mu and kappa1 opioid-stimulated [35S]GTPgammaS binding in tissue sections and membranes from cynomolgus monkey brain using DAMGO and U50,488H, respectively. Concentrations of agonists required to produce maximal stimulation of [35S]GTPgammaS binding were determined in membranes from the frontal poles of the brain. Receptor specificity was verified in both membranes and sections by inhibiting agonist-stimulated [35S]GTPgammaS binding with the appropriate antagonist. Mu opioid-stimulated [35S]GTPgammaS binding was high in areas including the amygdala, ventral striatum, caudate, putamen, medial thalamus and hypothalamus. Dense mu-stimulated [35S]GTPgammaS binding was also found in brainstem nuclei including the interpeduncular nucleus, parabrachial nucleus and nucleus of the solitary tract. Kappa1 opioid-stimulated [35S]GTPgammaS binding was high in limbic and association cortex, ventral striatum, caudate, putamen, globus pallidus, claustrum, amygdala, hypothalamus and substantia nigra. These results demonstrate the applicability of [35S]GTPgammaS autoradiography to examine receptor-activated G-proteins in the primate brain and reveal functional mu and kappa1 opioid receptor activity that may contribute to the reported central nervous system effects of opiates.